Pluripotent vaccine against enveloped viruses

ABSTRACT

The present invention is directed to compositions and methods for the induction of immune responses in mammals against enveloped animal viruses. More particularly, the invention provides vaccine compositions containing multiple MHC allotypes. By generating an immune response against these MHC molecules, virus or virus-infected cells expressing foreign MHC molecules can be attacked prior to infection of cells in the immunized host. In some embodiments, the vaccine compositions contain viral antigens and adjuvants as well. The vaccine compositions may comprise intact cells, cell-derived membrane preparations or recombinantly or chemically produced MHC molecules or fragments thereof.

BACKGROUND OF THE INVENTION

[0001] I. Field of the Invention

[0002] The field of the invention relates to compositions and methodsfor the induction of immune responses against enveloped virus inmammals. In particular embodiments, the invention relates to vaccinescomprising major and minor histocompatibility complex antigens, bloodgroup antigens and other cell surface antigens exhibiting significantgenetic polymorphism in mammalian populations, which may be picked up byenveloped viruses as they bud from the cell membrane and may thereforeserve as a target for an immune attack.

[0003] II. Description of the Related Art

[0004] A. Enveloped Viruses

[0005] Eukaryotic viruses are a large and diverse group of infectiousagents primarily known for the diseases they cause. These agents can beclassified by a number of criteria including genome structure, mode ofreplication and host specificity. Another manner of grouping eukaryoticviruses is by the structure of the viral particle. “Non-enveloped” viralparticles are made up of a proteinaceous capsid that surrounds andprotects the viral genome. The capsid is formed by viral structuralproducts encoded by the virus genome and synthesized within the hostcell. “Enveloped” viruses also have a capsid structure surrounding thegenetic material of the virus but, in addition, have a lipid bilayer“envelope” that surrounds the capsid. The envelope is acquired as thecapsid buds through one of the host cell membranes—usually the plasmamembrane but sometimes from the nuclear membrane, the Golgi apparatus orendoplasmic reticulum.

[0006] Exemplary enveloped virus families include Togaviridae,Flaviviridae, Coronaviridae, Rhabdoviridae, Filoviridae,Paramyxoviridae, Orthomnyxoviridae, Bunyaviridae, Arenaviridae,Retroviridae, Herpesvindae, Poxviridae and Iridoviridae. These virusesand others are responsible for such diseases as encephalitis, intestinalinfections, immunosuppressive disease, respiratory disease, hepatitisand pox infections.

[0007] The make-up of an enveloped virus membrane varies depending onthe location in the host cells from which the virus acquired itsenvelope. In general, the envelope comprises a bilayer of lipidscompletely surrounding the virus capsid or nucleoprotein. In addition tovarious lipids, the envelope contains integral and transmembraneproteins. Many transmembrane proteins have sugar residues attached andare referred to as glycoproteins. Virally-encoded transmembrane proteinsplay important roles in the infectious process by acting as targetingligands, as enzymes and as membrane fusion activators. Because hostcells also express many membrane-bound proteins, it is possible forenveloped virus particles to contain host cell proteins as well as thosethat are virally-encoded. In some instances, the virus may pick up genesof normal cellular components from the host cell which are useful to itspropagation in other hosts.

[0008] Considerable efforts have been expended toward the development ofsuitable vaccines designed to protect against infection bydisease-causing enveloped viruses. Though some vaccines have provedsuccessful, many others have failed to live up to expectations. Vaccinefailures often are attributed to one or more of the following:

[0009] 1. Enveloped viruses have a notorious reputation for a phenomenoncalled “antigenic drift.” When antigenic drift occurs, viral antigensmutate and their antigenic profile is altered. If the drift is extensiveenough, the immune response generated against the original antigenicprofile is no longer able to recognize the mutated forms and, therefore,escape the protective immune mechanisms within the immunized host.

[0010] 2. Another type of “variation” problem relates to viruses havingmultiple strains, such as rhinovirus, which may have more than 50different antigenic strains. Therefore, it is impractical to developvaccines against all of these viral strains. Yet another example ofantigenic variation is exhibited by influenza viruses, which frequentlyhave seasonal variations in the prevailing strains. Thus, it isnecessary to redesign particular influenza vaccines on an annual basis,depending upon the prevalent strain of influenza virus that is infectingthe population that particular year.

[0011] 3. Unfortunately, cell mediated immunity or humoral antibodyinduced against virus-related envelope or nuclear protein antigens maybe short-lived. As a result, most current vaccines are capable ofinducing immunity only for a short time following immunization. Thus, toachieve ongoing protection, it often is necessary to immunizerepeatedly, especially where the antigen is the inactivated virus or asubunit vaccine.

[0012] 4. A problem with many viral vaccines is the cost-benefitanalysis of choosing a “live” versus a “killed” vaccine. In general,immunization with live, attenuated viruses results in a much strongerimmune response than with killed virus. In some instances, such asprotection against smallpox maybe life long after immunization with livecowpox virus which induces cross reactive immunity to smallpox. Whilethe mechanism behind this phenomena is not fully understood, it islikely that limited replication of attenuated viruses provides a morepotent set of antigens with which to stimulate the immune system.Unfortunately, virus strains with sufficient attenuation levels aredifficult to produce. In addition, one always runs a risk that theattenuated virus will revert to a pathogenic form followingimmunization, thereby causing full-blown infection and disease.

[0013] Because of many problems outlined above with respect to viralvaccines, effective vaccines have been developed against only a smallminority of the many viruses that are capable of infecting the humanpopulation. Efforts have been directed primarily against viral vaccinescausing potentially fatal illness, such as smallpox. Consequently, thereare but a small number of effective vaccines developed against the largenumber of enveloped viruses which create diseases in humans and othermammals. Although there have been a few successes, such as vaccinesagainst smallpox, measles, mumps, feline leukemia virus and caninedistemper virus, humans and other mammal species remain largelyunprotected against the vast majority of enveloped viruses.

[0014] B. HIV and MHC Antigens

[0015] Because of the devastating consequences of infection, and therapidly growing number of infected individuals, there have been intenseefforts directed at producing a vaccine against human immunodeficiencyvirus (HIV) infection. Because of the danger in using live retroviruses,these vaccines primarily have involved the use of viral subunits, suchas the surface glycoprotein gp120/160, and antibodies thereto. For themost part, the results have been disappointing.

[0016] A variety of unique problems are presented when working with HIVvaccines. For example, the antigenic drift seen in HIV antigensvirtually is unparalleled in other systems. In addition, the putativeidentification of the CD4 molecule as a receptor, while constituting amajor step towards understanding the virus life cycle, has proved to bea problematic complication. Interactions between host molecules and CD4,as well as those between host molecules and gp120/160, appear to hinderthe effects of anti-gp120/160-based vaccines.

[0017] Another disadvantages in using specific immunization against HIVis that one of the primary methods for detecting infection is by thepresence of serological reactivity with HIV in the serum of theindividuals, indicating that infection with HIV has occurred.Consequently, immunization with HIV or antigenic subunits thereof willinduce similar antibodies, making it difficult to differentiateinfection from protection.

[0018] One area of research that has developed in response to these andother problems involves the use of major histocompatibility (MHC)antigens. The general principle behind this work is that an infectingHIV particle, either as free enveloped virus or as virally-infectedcells, will be associated with MHC antigens that are potentiallydistinct from those of the recipient. The literature as a whole is, atbest, confused with respect to how such an immune response mightfunction if, in fact, it would function at all.

[0019] For example, Clerici et al (1990) discuss the use of alloantigenimmunization to bolster the flagging T-helper functions in HIV patients.Clerici, as had others, observed that despite the inability of the Tcells of AIDS patients to “recall”antigens, these cells remainedresponsive to alloantigens. Clerici found that when the alloantigenresponse was still present, T cells of HIV⁺ individuals could be“taught” to recall influenza antigens when primed with influenza antigenin combination with alloantigen and challenged with influenza antigenalone. The ability of alloantigen alone to encourage recall of influenzaantigen by T cells in AIDS patients was negligible, suggesting acontribution by the influenza antigen in the role of both a stimuli anda target.

[0020] Another study that looked at the effects of cellular antigens(xenoantigens) on responses to immunodeficiency viruses was reported byStott (1991). Stott and his colleagues observed that macaques immunizedwith uninfected human cells were resistant to infection by simianimmunodeficiency virus (SIV) grown in those cells. Macaques were notresistant to infection by SIV grown in simian cells. When the challengevirus was HIV, the immunized macaques were not resistant, suggestingsome correlation of protection with the nature of the viral antigen.Stott also identified the anti-MHC response as directed against Class IIdeterminants in the human cells but it is unclear whether these werespecies specific or xenogenic antigenic determinants of class II pickedup by the viruses grown in human cells or truly cross-reactive allotypicantigens also present in the monkey.

[0021] Kion and Hoffmann (1991) demonstrated a similar phenomenon inalloimmunized mice. When immunized with cells from another murinestrain, mice made antibodies against gp120 and p24 of HIV. In addition,an autoimmune mouse strain made antibodies against gp120. In bothalloimmune and autoimmune mice, antibodies against anti-MHC antibodies(MHC-image) were found. The authors concluded that the presence of boththese kinds of antibodies “supports the idea of synergy between immuneresponse to allogeneic cells and HIV antigenic stimuli.”

[0022] Langlois et al. (1992), using SIV grown in human cells, were ableto elicit responses against human cellular determinants in macaques.Cranage et al. (1992) also reported that human-specific responsesdevelop when macaques are immunized with SIV grown in human cells. Whileinitially reporting that the predominant response was against class Iantigens, later studies were able to find no correlation between class Ititers and protection. Cranage et al. (1993) reported that macaquesvaccinated with SIV grown in human cells generate a class II-specificresponse not seen in control animals. Interestingly, infected macaqueswere shown to have antibodies specific for rhesus MHC, raising thepossibility that SIV mimics rhesus MHC or alters macaque recognition ofself-MHC.

[0023] Kiprov et al. (1994) took advantage of their earlier, unrelatedstudies on alloimmunization to look at anti-cellular/anti-HIV response.A group of women previously had been immunized with peripheral bloodlymphocytes from their husbands in an effort to prevent spontaneousabortion. Twelve out of fourteen women immunized developedantilymphocyte antibodies (ALAs). Of these, two were found to neutralizeHIV-1 in vitro in a complement dependent manner, one patient to arelatively high titer. Because addition of anti-HIV sera resulted inadditive neutralization, Kiprov suggested that the test sera wasdirected to nonviral target antigens. The authors could not explain theabsence of neutralizing activity from the other ALA+ subjects, however,and the mechanism for high titers of neutralizing antibodies in the onepatient remains unexplained since subsequently it has been shown not tobe related to MHC class I or II antigens.

[0024] Thus, it remains unclear whether MHC stimulates the immune systemin a non-specific manner, or specifically increases immune responsesagainst viral targets. It also is unclear whether xeno or alloimmuneeffects on SIV/HIV are, in any way, attributable to the MHC-likecharacter of gp120 and thus limited by the particular viral antigen.Thus, despite considerable speculation and some interesting results withan SIV model, the role played by alloimmunity in antiviral defenseremains unclear.

[0025] For all the above reasons, there is a great need for moreefficient viral vaccines against enveloped viruses. When one considersall of the possible antigenic strains and frequency of antigenic driftof various viruses which may affect various mammalian species, it isimpractical with present methodology to comprehend a universal viralvaccine which has as it target the viral antigens themselves. On theother hand, a vaccine which has as its target cellular membrane antigenswhich are expressed by genes controlling genetic diversity in the animalspecies could be pluripotent. The enveloped viruses must pick upgenetically controlled antigens from the cell membrane as they bud fromthe host cell which provide a much more stable and practical target forthe induction of protective immunity against membrane viruses. Thealloantigens which the enveloped viruses pick up when they bud from thehost cell membrane provide a potential target in the development of apluripotent viral vaccine protective against the majority of theseviruses.

III. SUMMARY OF THE INVENTION

[0026] It is, therefore, an object of the present invention to provide acomposition suitable for administration to a subject that inducesprotective immunity to infection by an enveloped virus.

[0027] It is another object of the present invention to provide acomposition suitable for administration to a subject that inducesprotective immunity to infection by human immunodeficiency virus.

[0028] It is yet another object of the present invention to provide amethod of immunizing a subject with a composition that inducesprotective immunity to infection by an enveloped virus such asrhinoviruses or influenza viruses.

[0029] It is still yet another object of the present invention toprovide a method of immunizing a subject with a composition that inducesprotective immunity to infection by human immunodeficiency virus.

[0030] In satisfying these objectives, there is provided a compositioncomprising intact cells, wherein said cells express majorhistocompatibility antigens with at least four common allotypes from agiven mammalian species.

[0031] There also is provided a composition of claim wherein saidallotypes each are present in 80% or more of individuals. There furtheris provided composition wherein any given cell expresses only a singleallotype.

[0032] In another embodiment, there is provided a composition wherein atleast one cell expresses at least two allotypes. There also is provideda composition wherein said antigens are Class I antigens, and there isprovided a composition wherein said antigens are Class II antigens.

[0033] In yet another embodiment, there is provided a compositionwherein said antigens are both Class I and Class II antigens or otheralloantigens coded by polymorphic genes. There further is provided acomposition wherein said plurality is representative of all knownallotypes of said mammalian species. Further embodiments include acomposition further comprising at least one recombinant major or minorallotypic antigen for the mammalian species. And in yet a furtherembodiment, there is provided a composition this recombinant antigen isproduced in a host selected from the group consisting of bacteria,fungi, insect cells and mammalian cells.

[0034] In a further embodiment the allotypes of the composition includeat least one of the following human allotypes:

[0035] HLAA₁, A₂, A₃, A₁₁, A₂₄, A₂₉, A₃₂,

[0036] B₇, B₈, B₁₃, B₃₅, B₃₈, B44, B₅₅, B₆₀, B₆₂,

[0037] CW₁, CW₂, CW₄, CW₅, CW₆, CW₇, CW₉, CW₁₀, CW₁₁,

[0038] DR₁, DR₃, DR₄, DR₇, DR₈, DR₁₁, DR,₁₂, DR₁₃, DR₁₅,

[0039] ABO Blood Groups.

[0040] In still yet another embodiment, there is provided a compositionwherein said cells further express an antigen from an enveloped virus.In further embodiments, the virus is a herpesvirus and/or a retrovirus.And in still yet further embodiments, the composition comprises intactcells further express a cytokine. The cytokine can be IL-1,IL2,IL-4,IL-7, IL-12, γ-interferon or GMCSF. Other embodiments provide intactcells further expressing a costimulatory molecule. The costimulatorymolecule is may be B-7.

[0041] In another embodiment, the cells of the composition are renderedincapable of growth; this may be accomplished by lethal irradiation. Thecomposition further may comprise a pharmaceutically acceptable carrier,diluent or excipient.

[0042] In an alternative embodiment, there is provided a method forgenerating an immune response in a given mammal comprising:

[0043] (a) providing a composition comprising

[0044] (i) intact cells, wherein said cells express majorhistocompatibility antigens with at least four allotypes from thespecies of said given mammal; and

[0045] (ii) a pharmaceutically acceptable carrier, diluent or excipient,

[0046] (b) administering said composition to said given mammal.

[0047] In another alternative embodiment, there is provided a method foreliciting an immune response in a given mammal against an envelopedvirus comprising:

[0048] (a) identifying a given mammal at risk of infection with saidvirus;

[0049] (b) providing a composition comprising

[0050] (i) intact cells, wherein said cells express majorhistocompatibility antigens with a plurality of allotypes from thespecies of said given mammal;

[0051] (ii) a pharmaceutically acceptable carrier, diluent or excipient,

[0052] (b) administering said composition to said given mammal in anamount effective to elicit said immune response.

[0053] In still yet another alternative embodiment, there is provided acomposition comprising intact, non-malignant cells, wherein said cellsexpress major histocompatibility antigens with a plurality of allotypesfrom a given mammalian species.

[0054] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

[0055]FIG. 1 provides a list of HLA allotypes and their frequency ofdistribution by ethnic groups.

V. DETAILED DESCRIPTION OF THE INVENTION

[0056] For every cell that is present in a mammal, that mammal mustdetermine whether that cell is “self” or “non-self.” If “self,” themammal does not want to mount an immune response. If non-self, themammal (host, in this case) usually wants to respond and eliminate thecell. A host further distinguishes two subsets of non-self cells—cellsthat the host has seen previously and cells that are new to the host.Biological mechanisms have evolved that permit a rapid and moresubstantial response to cells that previously have been encountered bythe host.

[0057] Thus, when a virus or virally-infected cell is exposed to a hostfor the first time, it is unlikely that a significant immune responsewill be mounted against the infectious agent before infection of hostcells occurs. In contrast, a host immune system that has been “primed,”i.e., made sensitive to a particular virus or viral antigen, can mount arapid and significant immune response that will prevent or limitinfection. This is the rationale behind vaccines, which are designed tosensitize the immune system of the host to one or more antigens of agiven virus. The following is a detailed explanation of the immuneresponse as it applies to the instant invention.

[0058] A. Immunity to Viruses Via Recognition of Antigen and Alloantigenby Antibody and by T Cells

[0059] Major and minor histocompatibility complex (MHC) glycoproteinswere studied intensively for many years without investigators having agood understanding of their function. These cell-surface antigens,exhibiting a high degree of genetic polymorphism in human and othermammalian populations, were subject to analysis using alloantibodiesraised by tissue immunizations between individuals of the same species.Since they also were major targets for specific immunological rejectionof transplanted tissues and organs and, in the absence of a specifiedfunction, they were called “transplantation antigens.”

[0060] Transplant rejection involves the recipient's T lymphocytes (Tcells) and B lymphocytes (B cells) responding to antigenic determinantscaused by structural differences in the MHC molecules of the donor andrecipient. Such determinants are called alloantigenic determinants, or“allodeterminants,” and the responding T cells are termed“alloreactive.” In the 1970's, cellular immunologists discovered thephysiologic function of MHC molecules, showing that they wereinstrumental in stimulating T cell responses to all antigens, not justalloantigens. Continuing study through the 1980's provided a mechanisticunderstanding of this relationship facilitated by the discovery that α/βand γ/δ T cell receptors, unlike B cell receptors (immunoglobulins), donot recognize native proteins but, rather, require that they be unfoldedand broken down into small fragments. This antigen “processing” takesplace within cells and, as far as is known, exploits enzymes andintracellular compartments that are, in general, used for the transport,turnover, maturation and processing of normal cellular proteins.

[0061] Thus, the role of MHC molecules is threefold: (1) to bindpeptides within the cell; (2) to transport them to the plasma membrane;and (3) to retain them at the cell surface in a complex which caninteract with the receptors of the T lymphocytes. The ligand recognizedby a T cell receptor is thus a complex of a peptide, usually 8-25 aminoacids in size, bound to an MHC molecule. The MHC glycoproteins are saidto “present” the peptide to the T cell and are aptly described as“antigen-presenting” molecules. Townsend and Bodmer, Ann. Rev. Immunol.7:601-624 (1989).

[0062] Two types of protein antigens can be presented by healthymammalian cells—those within the cell (e.g., viral proteins made afterviral infection of the cell) and those made elsewhere which then enterthe cell by endocytosis (e.g., bacterial toxins). Two homologous classesof MHC molecules have evolved as specialized with regard tointracellular movement and antigen presentation. Class I MHC moleculesprimarily present peptides derived from endogenously made proteins suchas those induced by intracellular viral infection, while class II MHCmolecules specialize in the presentation of peptides derived fromendocytosed antigens. Although this is the most common situation, therecan be exceptions in which the conditions of antigen presentation by MHCmolecules are reversed.

[0063] Two types of T cells are thought to interact with the two classesof MHC molecules. CD4 lymphocytes are helper T cells thought to receiveinstructions from the MHC class II antigen, which presents processedantigen peptides in the MHC class II grove. On the other hand, CD8lymphocytes (cytotoxic T cells) recognize antigenic peptides bound toMHC class I antigens. Antigen processing also occurs in B cells, whichcan endocytose and fragment the antibody/antigen complex. This complexis formed when the antigen reacts with B cell receptor, membrane-boundimmunoglobulin antibody directed against the antigen. In addition,macrophages can endocytose antigen-antibody complexes via F_(c)receptors, leading to enhanced processing and presentation to T cells.It is important to emphasize that in order for the T cell to recognizean antigen presented by either a class I or class II MHC molecule, theremust be a direct HLA match between the lymphocyte and the cellpresenting the antigen or there must be recognition of a cross reactingepitope on the MHC molecule by the T cell receptors of the T cell. Thus,a cytotoxic T cell of MHC class I A2 usually cannot recognize and kill acell bearing the same antigen which has been processed and presented byMHC class I A1 human cells.

[0064] It is important to emphasize that the two arms of the immunesystem (humoral and cell-mediated) recognize antigen in different forms.Antibody, produced by B cells, recognizes predominantly conformationalepitopes on the surface of large antigen molecules. T cells, via theirantigenic specific receptor, recognize linear sequences of peptidesproduced by processing of antigens by enzymatic breakdown of the nativeprotein. Thus, most T cell epitopes are not recognized in proteins intheir natural state and must be actively created, either during proteinsynthesis (i.e., before the molecule has folded into its correct finalshape) or by a reductive enzymatic process that involves some form ofprotein degradation. T cell epitopes are therefore generally independentof the native conformation (secondary and tertiary structure) and, thus,their recognition invariably has less discriminatory power than B cellepitopes which depend upon surface conformational structures. However,antigen processing exposes the immune system to a set of structures thatare normally buried within the native confirmation of macromoleculesand, therefore, would otherwise be invisible to immune recognition. Thisdouble ability to recognize both internal structure by T cell receptorsand external configuration by B cell antibodies is integrated into theoverall protective scheme of the immune system.

[0065] The second major difference between the two arms of the adaptiveimmune system is that T cell recognition does not occur with antigenalone. The two molecules, T cell receptor and epitope, only interact onthe surface of a separate cell, the antigen processing cell. Further,this cell is required to express a third molecule, either class I orclass II, of the major histocompatibility complex glycoprotein. Thesecell-surface glycoproteins have been shown to directly bind T cellepitopes. This binding step is the essential screening process by whichappropriately processed antigen is selected from the unprocessedmajority. The majority of evidence suggests that correct T cellrecognition can only occur if the appropriate antigen epitope ispositioned within a relatively small molecular groove at the distal endof a MHC molecule.

[0066] Thus, the molecular and cellular mechanisms by which T cellepitopes are created from complex macromolecules, and are then expressedby antigen presenting cells in a form that can be recognized together,constitute antigen processing. Antigen presentation is defined as thesubsequent interaction between antigen presenting cell and T cell in thepresence of peptide antigen. Therefore, antigen processing is a crucialstep in the overall series of events leading to T cell stimulation and,hence, in the development of an effective immunological response.

[0067] B. Two Pathways of Antigen Processing

[0068] The presence of two separate classes of MHC molecules on thesurface of cells has long been known. This class division is related tothe separation of mature T lymphocytes into two distinct groups. Themutually exclusive T cell surface antigens CD8 and CD4 providespecificity for MHC classes I and II, respectively. In contrast, it hasbeen shown that antigen-specific T cell receptors use the same pool ofgermline elements for recognition of antigen bound to both classes ofMHC. The class I-associated antigens are derived predominantly frominternal antigens (such as viral antigens) synthesized endogenously bythe antigen processing cells, while class II-associated antigenstypically are external, acquired exogenously by endocytosis from theextracellular environment. MHC class I antigens can be expressed in allnucleated cells. Therefore, via the class I processing pathway, theimmune system can recognize and react to a whole array of intracellularantigens that, in their native state, may never be exposed to theextracellular environment. This is consistent with the function of CD8+T cells, to scan all cells in the body for viral antigens that may bepresented with MHC class I molecules, thereby protecting the host fromintracellular viral infection. MHC class II antigens are found mainly onspecialized cells within the immune system, in particular B cells,macrophages, and dendritic cells. See Critical Reviews in Biochemistryand Molecular Biology, 26:439-473 (1991) by T. P. Levine and B. M.Chain, “The Cell Biology of Antigen Processing.”

[0069] C. Viral Infection and Implications for Immunity AgainstEnveloped Viruses

[0070] A viral protein antigen thus may be broken down into differentpeptides which are presented as distinct peptide sequences in the grooveof different MHC molecules, depending upon the genetics of host's MHCallele expression. Processing and presentation are key steps in thedevelopment of an effective immune response.

[0071] Transmission of envelope viruses may occur by infection with thefree viral particle itself, such as occurs by aerosol transmission ofrespiratory viruses like rhinoviruses or influenza virus due to coughingor sneezing by affected individuals. In addition, transmission can occurby transfer of infected cells, such as transfer of HIV-infectedlymphocytes via the semen. These cells may carry the HIV viral genome,yet little or no free virus may be present at the moment oftransmission. Although viral related peptide T cell epitopes may beexpressed by the MHC class I molecules on the surface of the infectedcell, these viral peptides cannot be recognized by the T cell receptorsof a recipient host unless there happens to be an allotype match betweenthe MHC class I or II of the donor and recipient. Thus, even though therecipient may already have ongoing long-term T cell memory againstpeptides associated with the infecting virus, this memory will beineffective against the peptide sequences presented by the MHCglycoprotein molecules on the incoming cell surface or viral membranebecause they cannot be recognized by the host T cells given the MHCmismatch.

[0072] The foreign protein viral antigens may be picked up by MHC classII antigen presenting cells of the host and presented to the CD4 helpercells. In addition, once the virus gains entry into the host's cells andbegins to replicate and synthesize proteins, the new host's MHC antigenswill present these different peptide sequences to cytotoxic T cells,which then act to inhibit further viral proliferation by killing theviral infected cells in the new host. This type of specific viralimmunity does not, however, prevent transmission of infection either byfree viral particles or infected cells, although it may limit the degreeof viral replication in the new host.

[0073] On the other hand, preexisting antibody directed against antigenson the surface of the viral particle may react directly with the viralparticle and prevent the initial infection. It is our hypothesis thatthe presence of preexisting allotypic antibody or T cell immunityagainst the predominant HLA antigens may trigger an immediaterecognition of the foreign alloantigens present on the enveloped lipidmembrane of the invading virus which, in turn, may trigger an immediaterecognition and attack of the foreign virus if the level of immunity issufficiently high. If low level immunity is present, it may triggerrecognition of the foreign alloantigen which would lead to cytokinerelease, accelerated specific antigen processing and rejection of thevirus via induction of specific immunity. This is an example of asecondary “helper” or bystander effect in the induction of specificimmunity which may inhibit the growth of virus in the new host to apathogenic level.

[0074] D. Viruses As Tissue Transplants: Possible Role in Evolution ofthe Allograft Response and Immunity to Enveloped Viruses

[0075] Organs can be freely transplanted between humans that areidentical twins or within strains of laboratory rodents where genetichomogeneity essentially eliminates the problem of diversity at the majorhistocompatibility complex. However, where genetic differences exist dueto diversity in HLA class I antigens at the A, B, and C locus, there israpid rejection of transplanted tissues by the new host due to a strong,potent and long-lasting allograft response. Since organ transplantationdoes not naturally occur in nature, it is tempting to hypothesize thatthe origin of this allograft response arose as an evolutionary mechanismto protect against allogeneic invasion by external agents. In otherwords, the MHC is a code that determines what is self and what isnon-self; non-self should be attacked and destroyed. One kind ofnatural, non-self, MHC-bearing agent would be enveloped viruses.

[0076] Genetic stability of indigenous native peoples in primitivetribes is considerably greater than that seen in urban populations.Thus, limited HLA diversity of native peoples would be expected. Whereviral infection occurs between individuals within the tribe, survivorswill gain the ability to respond against the tribes limited repertoireof MHC antigens, in addition to the viral antigens. Eventually, thiskind of limited exposure would permit development of alloreactive T cellresponses against all the MHC allotypes present in that tribe. Thesestrong alloreactive T cell responses against a limited number ofalloantigens would be as effective, or perhaps even more effective, thanimmunity directed against the specific viral epitopes. At a minimum,this kind of response benefits from its universality, i.e., it is notvirus specific. Moreover, in those situations where there was by chancean MHC class I match between the infecting envelope virus and the newhost, the peptide sequence presented in the MHC groove of the envelopevirus would be more rapidly recognized by the cytotoxic T cells of thenew host and thus trigger a rapid T cell response.

[0077] As modern, urban populations were formed by migration ofindividuals from many different tribes, the diversity of MHC antigens ina given population increased dramatically. It was impossible forisolated tribes to develop allotypic immunity against such large numbersof different HLA types given the lack of exposure. When brought intocontact with larger civilizations, the chances of an infecting virus orvirus-infected cell carrying either (i) the same allotype as the smalltribe recipient or (ii) an allotype to which the small tribe recipienthad previously been exposed, was greatly reduced. Consequently, it isnot surprising how devastating were the viral epidemics that occurredwhen European sailors came in contact with isolated native peoples ofisland communities.

[0078] Thus, from an evolutionary standpoint, the strong allograftimmune response, which certainly did not evolve to protect humansagainst tissue transplantation, is more likely to have evolved as aprotection against infection from transfer of cellular antigens pickedup from the host cell by enveloped viruses. This may be thought of asnature's version of graft-versus-host rejection. As further support forthis hypothesis, it is not unusual to find naturally occurring anti-HLAantibodies to various loci in human males and females who have neverundergone transplant, blood transfusion or become pregnant. It ispossible that these antibodies were induced by infection with envelopedviruses having foreign HLA antigens.

[0079] Although the emphasis in this discussion has been on MHC class Igenes coded by the A, B, C loci, there are also less well characterizedD, E, and F alleles and class II MHC antigens consisting of HLA DR, DP,and DQ which might serve as targets for an alloimmune response. Inaddition, due to genetic diversity in populations, other membraneantigens that are under genetic control such as blood group antigens ofthe ABO and RH types could also conceivably serve as targets for analloimmunization against envelope viruses.

[0080] E. Rationale

[0081] Because enveloped viruses acquire host cell membranes as they aresynthesized, virus particles are expected to contain host cell MHCcomponents as well as other cell surface antigens coded by diverse genessuch as ABO blood groups. Intact infected cells will carry MHCcomponents and other polymorphic antigens as well. MHC components inviral particles and infected cells provide a class of antigens inaddition to viral antigens that could be the target of a protectiveimmune response. Again, however, an unprimed immune response againstforeign MHC likely would not be able to prevent infection. Thus, itwould be necessary to prime the host immune system to respond againstforeign MHC components and other alloantigens.

[0082] The present invention operates on the premise that, in fact, aneffective vaccine can be designed around the use of MHC antigens and,perhaps, other allotypic antigens, more particularly, cells expressingMHC antigens. By priming an individual to response to foreign MHCantigens, it is believed that each enveloped virus particle or virusinfected cell will be subject to a rapid and substantial immune responsewith activation of both the antibody mediated B cell and T cell arms ofthe immune response, and thereby prevent infection of host cells.

[0083] In its most basic form, the present invention is a vaccinecomprising a plurality of MHC allotypes. If the vaccine comprises asingle allotype, that vaccine will not stimulate any immune response inthose individuals having the same allotype. This is unlikely exceptbetween identical twins because of the great genetic diversity presentat the HLA A, B, and C allotypes, as well as, class II allotypes ofmodern civilization. Thus, a vaccine representing at least one or moreallotypes will be of some benefit to every individual. The MHC antigensthat make up various allotypes are expressed on the surface of intactcells or are part of membrane preparations derived from cells expressingMHC antigens. In addition, in some embodiments the vaccine also willcontain viral encoded antigens and/or adjuvants. The followingdiscussion will further describe the attributes of such vaccines andtheir use.

[0084] F. MHC Antigen Profiles

[0085] In order for an effective MHC-based vaccine to protect anindividual against all infecting virus particles, that vaccine mustprovide the full spectrum of MHC antigens. For humans, this would meanthat a single vaccine would have to include sufficient allotypes of MHCantigens to guarantee that at least one of the allotypes present on thevirus envelope would be perceived as foreign by the vaccine recipient.FIG. 1 provides a list of HLA allotypes and their frequency ofdistribution by ethnic groups. Statistical analysis will yieldappropriate combinations of antigens permitting maximal protection. Thiskind of vaccine would not be specific for a given virus.

[0086] In many cases, however, it is unnecessary for the vaccine torepresent all possible MHC allotypes. Rather, substantial benefit willbe achieved by use of a vaccine that contains antigens representing onlythe most common allotypes or allotypes that are regionally prevalent.For example, if an individual would be protected from virus arising froma majority of the population using only select allotypes, such a vaccinewould have significant utility and might cost far less to produce than avaccine with maximal efficacy, i.e., with antigens representative ofevery allotype. In fact, so long as more than one allotype isrepresented in the vaccine, the recipient of the vaccine will beimmunized against at least one other allotype than his or her own.Again, such a vaccine would induce protection regardless of the natureof the infecting virus. Thus, the present invention comprises vaccineshaving two, three, four, five, six, seven, eight, nine, ten or more MHCallotypes.

[0087] The allotypic antigens used according to the present inventioncan be any of the major or minor histocompatability antigens or bloodgroup antigens. In one embodiment, these antigens are provided in a cellfree form. Such antigens may be purified from appropriate cells sourcesor, preferably, are produced by recombinant means following cloning ofthe corresponding gene. Methods by which cloning of allotypic antigensmay be accomplished are well known to those of skill in the art. SeeFinney, “Molecular Cloning of PCR Products” in CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Ausubel et al. Eds., John Wiley & Sons, New York(1987), p. 15.7.1.

[0088] Once the entire coding sequence of an allotypic gene has beendetermined, the gene can be inserted into an appropriate expressionsystem. The gene can be expressed in any number of different recombinantDNA expression systems to generate large amounts of the polypeptideproduct. Examples of expression systems known to the skilledpractitioner in the art include bacteria such as E. coli, yeast such asPichia pastoris, baculovirus, and mammalian expression systems such asin Cos or CHO cells. In a preferred embodiment, polypeptides areexpressed in E. coli and in baculovirus expression systems. A completegene can be expressed or, alternatively, fragments of the gene encodingportions of polypeptide can be produced.

[0089] The gene sequence encoding the antigen is analyzed to detectputative transmembrane sequences. Such sequences are typically veryhydrophobic and are readily detected by the use of standard sequenceanalysis software, such as MacVector (IBI, New Haven, Conn.). Thepresence of transmembrane sequences is often deleterious when arecombinant protein is synthesized in many expression systems,especially E. coli, as it leads to the production of insolubleaggregates which are difficult to renature into the native conformationof the protein. Deletion of transmembrane sequences typically does notsignificantly alter the conformation of the remaining protein structure.

[0090] Moreover, transmembrane sequences, being by definition embeddedwithin a membrane, are inaccessible. Antibodies to these sequences willnot, therefore, prove useful in vaccines. Deletion oftransmembrane-encoding sequences from the genes used for expression canbe achieved by standard techniques. See Ausubel et al., supra, Chapter8. For example, fortuitously-placed restriction enzyme sites can be usedto excise the desired gene fragment, or PCR-type amplification can beused to amplify only the desired part of the gene. If these transgenesare to be used as part of a whole cell vaccine, however, retention ofthe transmembrane sequences is desired.

[0091] The gene or gene fragment encoding an can be inserted into anexpression vector by standard subcloning techniques. For example, an E.coli expression vector is used which produces the recombinantpolypeptide as a fusion protein, allowing rapid affinity purification ofthe protein. Examples of such fusion protein expression systems are theglutathione S-transferase system (Pharmacia, Piscataway, N.J.), themaltose binding protein system (NEB, Beverley, Mass.), the FLAG system(IBI, New Haven, Conn.), and the 6×His system (Qiagen, Chatsworth,Calif.).

[0092] Some of these systems produce recombinant polypeptides bearingonly a small number of additional amino acids, which are unlikely toaffect the antigenic ability of the recombinant polypeptide. Forexample, both the FLAG system and the 6×His system add only shortsequences, both of which are known to be poorly antigenic and which donot adversely affect folding of the polypeptide to its nativeconformation. Other fusion systems produce polypeptide where it isdesirable to excise the fusion partner from the desired polypeptide. Ina preferred embodiment, the fusion partner is linked to the recombinantpolypeptide by a peptide sequence containing a specific recognitionsequence for a protease. Examples of suitable sequences are thoserecognized by the Tobacco Etch Virus protease (Life Technologies,Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).

[0093] In a preferred embodiment, the expression system used is onedriven by the baculovirus polyhedrin promoter. The gene encoding thepolypeptide can be manipulated by standard techniques in order tofacilitate cloning into the baculovirus vector. A preferred baculovirusvector is the pBlueBac vector (Invitrogen, Sorrento, Calif.). The vectorcarrying the gene for the polypeptide is transfected into Spodopterafrugiperda (Sf9) cells by standard protocols, and the cells are culturedand processed to produce the recombinant antigen.

[0094] As an alternative to recombinant polypeptides, synthetic peptidescorresponding to the antigens can be prepared. Such peptides are atleast six amino acid residues long, and may contain up to approximately35 residues, which is the approximate upper length limit of automatedpeptide synthesis machines, such as those available from AppliedBiosystems (Foster City, Calif.). Use of such small peptides forvaccination typically requires conjugation of the peptide to animmunogenic carrier protein such as hepatitis B surface antigen. Methodsfor performing this conjugation are well known in the art.

[0095] G. Supplementing an MHC Vaccine with Viral Antigens and Adjuvants

[0096] One significant limitation exists, however, with respect to avaccine based solely on MHC antigens. As stated above, a mammalianimmune system is able to differentiate self from non-self cells and themechanism by which self/non-self distinction is made involves MHCantigens. Thus, where an infecting virus or virus-infected cell happensto have arisen in an individual whose allotype is similar to the newlyinfected host, the MHC antigens on the infecting virus or virus-infectedcell may not be seen as foreign and, hence, will not elicit an immuneresponse regardless of whether or not the newly infected host wasimmunized with self-MHC antigens. Thus, an additional immune mechanismis required to provide immunity against virus and virus-infected cellsarising from individuals of the same allotype.

[0097] One way in which this problem may be overcome is by addingantigens from the virus. Thus, where the infecting virus was generatedin an individual with the same allotype as the individual beinginfected, an additional non-self recognition mechanism will be provided.Unlike an MHC-directed response, immunization with viral antigens wouldresult in a immune response that was specific for a given virus.

[0098] Selection of viral antigens for use in supplementing theMHC-based vaccine of the present invention will be based on thefollowing considerations. First, the selected antigens should becommonly and stably expressed in the envelope of the virus and themembrane of infected cells for induction of protective immunity. Theseantigens will be accessible to the antigen processing cells of theimmune system of the individual being infected. Second, it is desirablethat the selected antigens are the immunodominant species for the virusin question. Third, it is preferred that the selected antigens do nothave any toxic or pathogenic function in and of themselves. Fourth,stable and immunodominant antigens presented by MHC class I complexfollowing endogenous processing may be most effective in limiting spreadof virus once infection of the new host has been successful. And fifth,it will be most expedient if the corresponding genes for the selectedantigens have been cloned.

[0099] In other situations, it will be desireable to provide adjuvantsthat enhance the immune response to allotypes that are perceived assimilar to, but distinct from, self. Such adjuvants include allacceptable immunostimulatory compounds such as cytokines, toxins orsynthetic compositions. Examples of these are IL-1, IL-2, IL4, IL-7,IL-12, γ-interferon, GMCSP, BCG, aluminumhydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thur-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmityol-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP) 1983A, referred to as MTP-PE), lipid A, MPL and RIBI, whichcontains three components extracted from bacteria, monophosphoryl lipidA, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%squalene/Tween 80 emulsion. The effectiveness of an adjuvant may bedetermined by measuring the amount of antibodies or cytotoxic T cellswith T cell receptors directed against an immunogenic polypeptidecontaining viral antigens resulting from administration of thispolypeptide in vaccines which are also comprised of the variousadjuvants. In some embodiments, it will prove beneficial to include bothviral antigens and adjuvants, along with MHC antigens, in a singlevaccine preparation. Where the adjuvants are polypeptides, it ispossible to include the genes for these polypeptides in a suitableexpression vector in a cellular version of the vaccine.

[0100] In addition to adjuvants, it may be desirable to coadministerbiologic response modifiers (BRM), which have been shown to upregulate Tcell immunity or downregulate suppressor cell activity. Such BRM'sinclude, but are not limited to, Cimetidine (CIM; 1200 mg/d)(Smith/Kline, PA); or low-dose Cyclophosphamide (CYP; 300 mg/m²)(Johnson/Mead, NJ) and Cytokines such as γ-interferon, IL-2, or IL-12 orgenes encoding proteins involved in immune helper functions such as B-7.

[0101] H. Cell Lines

[0102] As stated above, one problem with the use of subunit vaccines,comprised of either recombinant antigens or antigens isolated from anatural source, is that the antigens may have lost their “native”character. Usually, this change results from the loss of higher orderstructure. Because the factors affecting secondary and tertiarystructure of proteins are manifold, it is difficult, if not sometimesimpossible, to generate effective subunit vaccines. This may explain, inpart, why live, attenuated vaccines have generally proved more effectiveat generating high level responses and long-lived immunity.

[0103] In order to address this problem, the present invention relies,in one embodiment, on intact cells to carry the MHC antigens.Conveniently, appropriate cell lines can be selected that representmajor MHC allotypes. Various cell lines may be mixed together to achievethe necessary MHC profile. Regardless of the precise make-up, asignificant advantage should accrue with the use of intact cells as theMHC antigens will be expressed and presented to the host in their nativemilieu. In fact, such cells should be analogous to live, attenuatedvaccines.

[0104] It is conceivable that a single cell line can be geneticallyengineered to express multiple allotypes or, at least, multiple allelesof MHC antigens. It is a matter of routine skill for those in the fieldto clone genes corresponding to various allotypes of different MHCantigens, given the existing sequence homology of these molecules andknowledge of their tissue distribution. In this way, it is possible toincrease the number of allotypes expressed in a given vaccine withoutincreasing the number of cell types required. In fact, it may bepossible to engineer a single cell that expresses a sufficient number ofallotypes that significant protection is afforded thereby. This also isa way to generate a vaccine with higher levels of MHC expression or toprovide an allotypic profile that is not available from a readilypropagated cell line.

[0105] In certain embodiments, it will prove useful to treat the cellscomprising the vaccine so that they do not replicate within thevaccinated host. This may be accomplished by a variety of meansincluding irradiation, formaldehyde fixation, heating or freeze-thawing.Any other method in which the cells are rendered non-replicative, butleft intact will, in theory, be useful. A preferred embodiment isirradiation since it has the ability to prevent replication of theliving cells in the immunized person, yet the cells remain alive andcapable of presenting alloantigenic, as well as, specific viral relatedpeptide sequences as T cell epitopes appropriate for each of the MHCclass I allotypes.

[0106] Where viral antigens are included in the vaccine, it iscontemplated that a cell line is stably transformed with one or moreviral antigens or the antigens may be isolated from various sources andsimply mixed with the existing vaccine. In a preferred embodiment, animmunizing cell of the vaccine is stably transformed with an expressionvector comprising a regulatory region that is functional in the celloperably linked to one or more viral antigen genes. These antigens arebelieved to be more stable than coat protein antigens and are lesssusceptible to antigenic drift. As stated above, this will permitprotection of subjects where an infecting virus or virus-infected cellcarries the same MHC allotype as the subject being infected. Anadditional benefit also may arise from the expression of viral antigensby cells. In certain cells, the viral antigen will be proteolytically“processed” and expressed in the context of the cells MHC molecules.This MHC “presentation” of antigen is an important part of antigenrecognition and the use of living irradiated cells that temporarilysurvive in the individual being immunized and permit such presentationmay result in improved priming of the immune response.

[0107] In another embodiment, it is contemplated that the cells will betransformed with genes encoding immunostimulatory compounds such asIL-1, IL-2, IL-4, IL-7, IL-12, γ-interferon or GMCSF. These products mayact locally in enhancing the recognition of vaccine cells by the immunesystem. They also may provide a more systemic effect that enhances otheraspects of the immune response.

[0108] DNA sequences useful for the transformation of cells can berecloned from standard plasmids into expression vectors withcharacteristics that permit higher levels of, or more efficientexpression of the polypeptide encoded therein. At a minimum, this wouldrequire a eukaryotic promoter sequence which initiates transcription ofthe inserted DNA sequences. A preferred expression vector is one wherethe expression is inducible to high levels. This is accomplished by theaddition of a regulatory region which provides increased transcriptionof downstream sequences under appropriate stimulation. Expressionvectors may be integrative (retrovirus) or episomal (bovine papillomavirus).

[0109] Expression vectors are transferred into cells by any standard DNAtransfer techniques. Calcium phosphate transformation, protoplastfusion, lipofection or electroporation are the preferred mechanisms fortransfer of the vector into cells. In most situations, it will bedesirable to include a selectable marker gene when transforming cells.When grown under selective conditions, surviving cells are much morelikely to have taken up and expressed the gene of interest along withthe selectable marker. All above methods are well known in the art.

[0110] The DNA's used to transform host cells preferably comprise asequence that is optimized for expression of the encoded product in agiven cell. Thus, in some instances, it will be desired that thenucleotide sequence to be expressed is a cDNA, while in othersituations, it will be preferred to use genomic sequences. Degeneracy incodons for most amino acids means that nucleotide sequences other thanthe cDNA or genomic sequences for a given gene may encode the desiredpolypeptide.

[0111] It is possible that the use of MHC and viral antigens togetherwill prove to be the best mechanism for induction of a protective immuneresponse generally, and not merely a mechanism to ensure protection ofsubjects from virus or cells derived from individuals with similarallotypes. In such a case, it will be preferred that cell linesexpressing one or more viral antigens be produced for each allotypedesired in the vaccine. Alternatively, it may be possible to generatecells expressing multiple allotypes as well as viral antigens.

[0112] It also is contemplated that cell membrane preparations can beused as the vaccine substance. Because many MHC molecules are membranebound, the use of membrane preparations could be used as an alternativesource of MHC antigen that is delivered to the subject nor would be itbe expected to substantially alter the higher order structure of suchmembrane bound molecules. Furthermore, since many of the MHC antigenshave been cloned, the MHC molecules may also be produced by recombinantgenetic engineering techniques.

[0113] In theory, any cell type may be used. Preferred characteristicsfor cells include ability to grow well in tissue culture, exhibit highamounts of MHC class I and II antigens, easily transducible with viralantigen genes and susceptible to irradiation which will make themincapable of prolonged growth in the new host. They must be free fromextraneous adventitious viruses.

[0114] As a general proposition, cells are selected such that therepresentative HLA types of the target population will be present in thecells. Cells can be obtained by punch biopsies from patients in thetarget population and typed by standard protocols. Alternatively,malignant cells from patient biopsies within the target population maybe employed for their superior growth in culture. Another possiblesource of normal or malignant cells is a cell depository such as theAmerican Type Culture Collection (Rockville, Md.).

[0115] Selected cells lines are screened for adventitious infection byviral pathogens by standard assays (immunocytochemistry, electronmicroscopy, etc.). Cells are cultured using standard techniques adaptedto the particular cell lines. When sufficient numbers of cells areavailable, the cells are irradiated with about 10,000 to 15,000 rads toinactivate the cells, preventing replication following administration. Asuitable number of cells is between 8 and 10 million per administration.

[0116]FIGS. 1 through 3 provide lists of HLA allotypes and theirfrequency of distribution by ethnic group. It is a matter of routineexperimentation to identify the needed allotypes and to selectappropriate cells lines that provide the proper antigens. For mostpopulations, it is estimated that no more than three or four cell lineswill be required to provide a vaccine that encompasses 100% of the HLAantigen of the target population.

[0117] In a preferred embodiment, a vaccine according to the presentinvention comprises whole melanoma cells that have been irradiated. In aparticularly preferred embodiment, the melanoma cells include threehuman melanoma cell lines (M-10VACC, M-24VACC, and M-101VACC), whichwere selected from a series of melanoma cell lines after carefulexamination for the high expression of certain MHC antigens, grown andprepared for administration as described in Hoon et al. (1990),incorporated herein by reference.

[0118] I. Methods for Administration

[0119] Administration of vaccine compositions according to the presentinvention will be via any common route including oral, nasal, buccal,rectal, vaginal, or topical. Alternatively, administration will be byintradermal subcutaneous, intramuscular, intraperitoneal, or intravenousinjection. Vaccine compositions would normally be administered aspharmaceutically acceptable compositions that include physiologicallyacceptable carriers, buffers or other excipients.

[0120] The pharmaceutical compositions of the present invention areadvantageously administered in the form of injectable compositionseither as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. These preparations also may be emulsified. A typicalcomposition for such purpose comprises a pharmaceutically acceptablecarrier. For instance, the composition may contain about 100 mg of humanserum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike may be used. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to well known parameters.

[0121] Additional formulations which are suitable for oraladministration. Oral formulations include such typical excipients as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonateand the like. The compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders.

[0122] The term “unit dose” refers to physically discrete units suitablefor use in humans, each unit containing a predetermined-quantity of thevaccine composition calculated to produce the desired immune response inassociation with its administration, i.e., the appropriate carrier,route and treatment regimen. The quantity to be administered, bothaccording to number of treatments and unit dose, depends on the subjectto be treated, capacity of the subject's immune system to respond andprotection desired. Precise amounts of vaccine composition also dependon the judgment of the practitioner and are peculiar to each individual.Suitable dosage ranges are of the order of 0.001 to 10 mg of the activeingredient. Suitable regimes for initial administration and boostershots also vary, but are typified by immunization at day 0, 14, 28, andevery 6 to 12 months thereafter.

[0123] J. A Prototype Vaccine

[0124] A model vaccine according to the present invention is melanomacell vaccine, also referred to as “MCV.” This vaccine consists of threemelanoma cell lines that are known to contain effective concentrationsof MHC antigens capable of inducing responses against a variety of HLAallotypes which are representative of a large proportion of human MHCclass I genes. In addition, the MCV contains six melanoma associatedantigens, also referred to here as “MAA.” These MAA's, including threegangliosides (GD2, GM2 and O-acetyl GD3) and three protein antigens (alipoprotein M-TAA, M-fetal antigen and M-urinary antigen) have beendemonstrated to be immunogenic in melanoma patients. These antigens arelocated on the cell surface, and antibodies to them have been shown tobind with complement and kill melanoma cells in vitro. Morton et al.,Prolongation of Survival in Metastatic Melanoma After Active SpecificImmunotherapy With a New Polyvalent Melanoma Vaccine, Annals of Surgery,216:463-482 (1992). Immunization of patients with MCV containing theseantigens induces specific immune responses to the MAA.

[0125] A Phase II trial was undertaken to evaluate use of MCV inpatients with advanced metastatic melanoma. Patients receiving thisvaccine have survived significantly longer than patients previouslytreated with other regimens of immunotherapy or chemotherapy. Thevaccine was administered to patients for melanoma metastatic to regionalskin and subcutaneous sites (AJCC Stage IIIA) as well as to distantsites (AJCC Stage IV). Compared to previous trials, the new vaccine wassignificantly more effective at eliciting specific humoral andcell-mediated immune responses. Those patients who were treated with thenew polyvalent MCV and developed high levels of humoral antibody and/orcell-mediated immune responses exhibited prolonged survival compared tonon-responding patients.

J. EXAMPLES Example 1

[0126] Melanoma Cell Vaccine

[0127] The active specific immunotherapy protocol involves immunizationof subjects with irradiated whole cell MCV. MCV consists of three humanmelanoma cell lines (M-10VACC, M-24VACC, and M-101VACC), which wereselected from a series of melanoma cell lines after careful examinationfor the high expression of melanoma associated antigens, grown andprepared for administration as described in Hoon et al. (1990),incorporated herein by reference.

[0128] The HLA types of the three cell lines in the MCV are given inTable V. It will be noted that the cross reacting alleles for the Alocus are likely to be present in 120% of the Caucasian population andfor the B locus 70% of the population.

[0129] An outside laboratory screened the MCV for viral (HIV,hepatitis), bacterial and fungal infectious organisms. Equal amounts ofeach line were pooled to a total of 24×10⁶ cells in serum-free mediumcontaining 10% dimethyl sulfoxide and TABLE V HLA Types of MCV APPROX.FREQUENCY OF HLA TYPE IN CAUCASIAN CELL LINE HLA TYPE POPULATION M-10 A3 24.7% A 11 20.5% B 7 23.6% B 55 8.2% M-24 A 1 26.7% A 29 9.6% B 384.1% B 42 M101 A 2 48.6% A 29 9.6% B 44 25.5%

[0130] cryopreserved in liquid nitrogen. After cryopreservation, thecells were irradiated to 100 GY.

[0131] Prior to treatment, MCV was thawed and washed three times inmedia RPMI 1640. MCV was injected intradermally in axillary and inguinalregions on a schedule of every 2 wk for three times, then monthly forone year. For the first two treatments, MCV was mixed with tice strainBCG (8×10⁶ organisms). After one year, the immnunization interval wasincreased to every 3 months for four times, then every 6 months.Follow-up clinical and laboratory evaluations were repeated monthly.

[0132] The antibody response to melanoma cell surface antigens followingMCV immunization are evaluated by the indirect membraneimmunofluorescence (IMIF) assay, as described in Morton et al., Surgery64:233-240 (1968); Jones et al., J. Nat'l Cancer Inst. 66:249-254(1981).

[0133] Delayed Cutaneous Hypersensitivity (DCH) are measured byintradermal skin tests with MCV before and during therapy. One-tenth ofthe pooled MCV (2.4×10⁶ cells) are administered at a remote site on theforearm. After 48 hours, the average diameter of the induration isrecorded as the DCH response. The student t-test was used to compare theabsolute values of DCH from wk 0 to 4 and to 16.

[0134] General immunocompetence is evaluated by sensitization andchallenge to DNCB and response to common skin test antigens such asmumps and Candida. The responses to purified protein derivative (PPD)antigen to which the patient becomes sensitized as result ofimmunization with BCG in the vaccine serves as additional controls.Mixed Lymphocyte Tumor Cell Reaction (MLTR) is used to evaluate the invitro response to immunization. PBL's from weeks 0, 4 and 16 areisolated and cryopreserved. Assays are performed on cryopreservedlymphocytes to ensure reproducibility. Serial bleed PBL aresimultaneously thawed, washed, and resuspended in culture medium (RPMI1640 with 10% human AB serum (heat-inactivated) (Irvine Scientific,CA)).

[0135] MCV, when administered alone, is very well tolerated, withvirtually no significant toxicity when administered up to 5 years at3-month intervals. Mild erythema and itching are noted in the treatmentsites by a majority of patients. This is transient, lasting only 2-3days. About 15 % of patients report low grade fever of <99° F. for 12 to24 hr. A similar proportion of patients report mild fatigue on the dayor two following treatment with MCV alone. Myalgia and arthralgia arerarely reported. When mixed with BCG, ulcerations at the injection sitesare commonly seen.

[0136] Example 2

[0137] Induction of Antibodies Reactive with Influenza A and B

[0138] We have tested the coded sera from patients immunized with thisMCV for antibodies reactive in a blinded ELISA assay with influenza Aand influenza B using the preimmunization sera as a control. The dataare summarized in Table I. We have found that 81% of the patients afterimmunization with MCV showed increased serological reactivity by ELISAto influenza A of at least 0.5 antigen units and 38% had similarincreased serologic activity to influenza B. This increased activity isbelieved secondary to cross-reacting cellular antigens in MCV. TABLE IInduction of Antibodies Reactive with Influenza A and B PATIENT ELISAUNITS INITIALS BLEED DATE INFLUENZA A INFLUENZA B D. D. Oct. 15, 19921.78 4.86 D. D. Nov. 16, 1992 2.84 6.56 D. D. Dec. 21, 1992 3.67 6.89 R.S. Jul. 27, 1994 2.1 2.99 R. S. Aug. 25, 1994 3.0 2.72 R. S. Sep. 29,1994 3.16 2.72 R. K. Mar. 23, 1988 5.39 5.89 R. K. May 05, 1988 6.958.05 R. K. Aug. 25, 1988 5.74 6.73 C. G. Aug. 03, 1994 4.62 9.09 C. G.Sep. 15, 1994 8.10 5.50 P. O. Jan. 30, 1986 5.58 1.73 P. O. Feb. 27,1986 5.80 1.73 P. O. Jun. 19, 1986 5.80 1.58 C. C. May 22, 1989 3.1 1.32C. C. Aug. 14, 1989 3.23 1.31 C. C. Apr. 24, 1989 3.65 1.51 J. S. Aug.06, 1985 5.8 8.36 J. S. Oct. 29, 1985 6.37 8.39 D. H. Oct. 13, 1986 2.227.6 D. H. Nov. 14, 1986 2.32 9.04 D. H. Feb. 06, 1987 2.23 8.45 J. H.Jul. 08, 1992 8.80 6.82 J. H. Aug. 05, 1992 7.47 5.59 J. H. Oct. 29,1992 8.59 6.95 F. A. Nov. 26, 1986 6.84 3.94 F. A. Jan. 12, 1987 7.393.43 F. A. May 07, 1987 7.93 3.58 M. H. Sep. 24, 1985 6.84 13.9 M. H.Oct. 18, 1985 7.9 13.8 M. H. Feb. 25, 1986 7.6 13.6 P. R. Dec. 02, 19859.05 1.97 P. R. Jan. 06, 1986 9.96 2.69 P. R. Mar. 25, 1986 8.55 2.1 P.R. May 12, 1986 9.75 2.12 D. H. Jul. 17, 1985 10.3 6.50 D. H. Aug. 13,1985 12.0 7.12 D. H. Jan. 03, 1986 8.88 5.38 S. V. H. Sep. 09, 1985 3.021.74 S. V. H. Oct. 10, 1985 4.55 2.34 S. V. H. Feb. 10, 1986 3.74 1.90J. W. Apr. 09, 1993 2.59 1.01 J. W. May 18, 1993 3.25 1.04 J. W. Nov.16, 1993 3.30 0.75 I. B. Dec. 03, 1991 6.10 7.18 I. B. Jan. 10, 19927.45 7.11 I. B. Apr. 22, 1992 6.01 6.63

[0139] Example 3

[0140] Induction of Cytotoxic Antibodies Reactive with Cell SurfaceAntigens on the Surface of Peripheral Blood Lymphocytes Taken from aPanel of 106 Normal Donors

[0141] The sera from 10 of the 16 immunized patients formed sufficienttiters of antibodies to induce cytotoxicity to allogenic lymphocytesfrom at least 20% of the test population of 116 normal lymphocyte donors(Table II) when tested by a standard Terasaki assay for cytotoxicantibodies. Six of the 16 individuals or 38% formed antibodies that werecytotoxic to >50% of the normal donor lymphocyte population. Thefrequency of cytotoxic antibodies varied from 20% to 97% againstlymphocytes from individuals in the test panel.

[0142] Thus, in summary, it is clear that immunization with MCV wassuccessful in inducing antibodies reactive with cellular antigenspresent on influenza A and B viruses by ELISA and capable of inducinglysis of allogenic lymphocytes in 63% of immunized subjects. These sameantibodies which were capable of causing lysis of lymphocytes in thepresence of complement would be capable of causing lysis of the viralmembranes of enveloped viruses which contained the same MHCalloantigens. TABLE II Induction of Cytotoxic Antibodies Reactive withPeripheral Blood Lymphocytes taken from a Panel of 116 Normal DonorsCYTOTOXIC ANTIBODY TO PHERIPHERAL PATIENT SERUM BLOOD INITIALS CODEBLEED DATE LYMPHOCYTES * D. D. JWHLA-01 Oct. 15, 1992 0 D. D. JWHLA-17Nov. 16, 1992 0 D. D. JWHLA-48 Dec. 21, 1992 0 R. S. JWHLA-02 Jul. 27,1992 0 R. S. JWHLA-25 Aug. 25, 1992 0 R. S. JWHLA-40 Sep. 29, 1994 28%R. K. JWHLA-03 Mar. 23, 1988 0 R. K. JWHLA-26 May 05, 1988 0 R. K.JWHLA-39 Aug. 25, 1988 80% C. G. JWHLA-04 Aug. 03 1994 0 C. G. JWHLA-18Sep. 15, 1994 0 P. O. JWHLA-05 Jan. 30, 1986 0 P. O. JWHLA-19 Feb. 27,1986 0 P. O. JWHLA-46 Jun. 19, 1986 0 C. C. JWHLA-06 May 22, 1989 0 C.C. JWHLA-27 Aug. 14, 1989  6% C. C. JWHLA-38 Apr. 24, 1989 0 J. S.JWHLA-20 Aug. 06, 1985 15% J. S. JWHLA-45 Oct. 29, 1985 56% D. H.JWHLA-08 Oct. 13, 1986 0 D. H. JWHLA-28 Nov. 14, 1986 0 D. H. JWHLA-37Feb. 06, 1987 0 J. H. JWHLA-09 Jul. 08, 1992 0 J. H. JWHLA-29 Aug. 05,1992 0 J. H. JWHLA-36 Oct. 29, 1992 20% F. A. JWHLA-10 Nov. 26, 1986 56%F. A. JWHLA-21 Jan. 12, 1987 68% F. A. JWHLA-44 May 07, 1987 65% M. H.JWHLA-11 Sep. 24, 1985 0 M. H. JWHLA-22 Oct. 18, 1985 0 M. H. JWHLA-43Feb. 25, 1986 0 P. R. JWHLA-12 Dec. 02, 1985 0 P. R. JWHLA-30 Jan. 06,1986 0 P. R. JWHLA-35 Mar. 25, 1986 97% P. R. JWHLA-47 May 12, 1986 90%D. H. JWHLA-13 Jul. 17, 1985 0 D. H. JWHLA-23 Aug. 13, 1985 70% D. H.JWHLA-42 Jan. 03, 1986 87% S. V. H. JWHLA-14 Sep. 09, 1985 0 S. V. H.JWHLA-24 Oct. 10, 1985 0 S. V. H. JWHLA-41 Feb. 10, 1986 29% J. W.JWHLA-15 Apr. 09, 1993 0 J. W. JWHLA-31 May 18, 1993 0 J. W. JWHLA-34Nov. 16, 1993 70% I. B. JWHLA-16 Dec. 03, 1991 0 I. B. JWHLA-32 Jan. 10,1992 0 I. B. JWHLA-33 Apr. 22, 1992 30%

[0143] Example 4

[0144] Prevention of Upper Respiratory Infections and Chest “Colds” inPatients Receiving the Melanoma Vaccine

[0145] To determine whether these in vitro immunologic reactions had anyin vivo significance in regard to preventing infectivity with envelopeviruses, a questionnaire was prepared which was filled out by 53melanoma patients who developed 1 or more episodes of upper respiratoryor chest “colds” per year and who had been receiving immunotherapy withthe melanoma cell vaccine for 9 months or more (a length of time whichis necessary to adequately sensitize against MHC alloantigens) (TableIII). We wanted to judge whether this immunization had affected theincidence of upper respiratory infections or chest colds duringimmunotherapy versus their usual incidence of infections beforeimmunotherapy. It was found that these patients exhibited an average of1.74 respiratory infections per year before immunotherapy, compared to1.11 after immunotherapy. Twenty-eight (53%) of these individualsreported no change in their incidence of such illness, whereas 4/53 (8%)reported an increase in frequency of “colds.” However, 21 of the 53 orapproximately 40% of the individuals, particularly those who had 2 ormore upper respiratory infections per year, reported a diminution infrequency and severity of such illnesses which are usually caused byenveloped viruses such as rhinoviruses, influenza, parainfluenzaviruses. Some individuals, particularly those who had three or moreviral infections per year prior to TABLE III Subset of Subjects withThree or More Colds/Flu Per Year PATIENT BEFORE AFTER INITIALSIMMUNOTHERAPY IMMUNOTHERAPY CHANGE S. B. 4 1 −3 B. C. 3 0 −3 D. H. 6 2−4 J. H. 4 0 −4 M. M. 4 1 −3 M. M. 3 1 −2 M. N. 3 5 2 R. P. 3 3 0 L. S.3 0 −3 M. W. 3 1 −2 R. B. 5 1 −4 D. R. 5 3 −2

[0146] immunotherapy showed a dramatic reduction in the frequency ofcolds and bouts of flu. Of the 12 patients who reported experiencingsuch frequent colds, 10 (83%) reported a decrease in the incidence ofcolds, one person reported no change, and only one reported an increasein number of colds. For the group that experienced a decrease in coldsand flu, the average number of colds before immunotherapy was 4; theaverage number of episodes experienced after immunotherapy was 1. SeeTable IV. TABLE IV Summary of All Immunized Subjects NUMBER OF COLDS/FLUPER YEAR PATIENT BEFORE AFTER INITIALS IMMUNOTHERAPY IMMUNOTHERAPYCHANGE R. B. 5 1 −4 R. B. 2 0 −2 S. B. 4 1 −3 J. B. 1 1 0 S. C. 2 1 −1B. C. 3 0 −3 G. D. 1 1 0 D. H. 6 2 −4 J. H. 4 0 −4 R. H. 1 1 0 N. K. 1 0−1 L. L. 1 1 0 W. M. 1 0 −1 M. M. 4 1 −3 M. M. 3 1 −2 J. M. 1 0 −1 R. P.1 0 −1 M. R. 1 1 0 L. S. 3 0 −3 A. W. 2 1 −1 M. W. 3 1 −2 M. N. 3 5 2 B.S. 1 3 2 B. T. 1 1 0 C. W. 1 3 2 D. A. 1 1 0 G. B. 1 1 0 C. B. 1 1 0 P.B. 1 1 0 J. C. 1 1 0 B. D. 1 1 0 O. D. 1 1 0 T. W. D. 5 3 −2 T. H. D 1 21 C. D. 1 0 −1 S. E. 1 1 0 E. F. 1 1 0 S. G. 1 1 0 W. G. 1 1 0 M. H. 1 0−1 C. H. 2 2 0 D. H. 1 1 0 P. H. 2 2 0 R. L. 1 1 0 M. M. 1 1 0 M. M. 1 10 R. P. 3 3 0 D. R. 1 1 0 D. S. 1 1 0 J. T. 1 1 0 C. T. 1 1 0 M. V. 1 10 K. W. 1 1 0 MEAN: 1.74 1.11 −0.63

REFERENCES

[0147] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0148] Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Chapter 8.John Wiley & Sons (1990)

[0149] Cranage, M. et al. Symposium for Nonhuman Primate Models in AIDS(PUERTO RICO), Nov. 17-20, 1992, 10 pabstract no. 113

[0150] Cranage, M. et al. Symposium for Nonhuman Primate Models in AIDS(UNITED STATES), Sep. 19-22, 1993, 11 pabstract no. 27

[0151] Finney. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Chapter 15. JohnWiley & Sons, New York (1987)

[0152] Hoon et al. Cancer Research 50:5358-5364 (1990)

[0153] Jones et al. J. Nat'l Cancer Inst. 66:249-254 (1981)

[0154] Kion, Tracy A. and Geoffrey W. Hoffmann. Science, 253:1138-1140(1991)

[0155] Kiprov, D. D., et al. Science, 263:737-738 (1994)

[0156] Langlois, Alphonse J., et al. Science, 255:292-293 (1992)

[0157] Levine, T. P. and B. M. Chain. Critical Reviews in Biochemistryand Molecular Biology26:439-473 (1991)

[0158] Morton et al. Surgery, 64:233-240 (1968)

[0159] Morton et al. Annals of Surgery, 216:463-482 (1992)

[0160] Stott, E. J. Nature, 353:393 (1991)

[0161] Townsend and Bodmer. Ann. Rev. Immunol. 7:601-624 (1989)

What is claimed is:
 1. A composition comprising intact cells, whereinsaid cells express major histocompatibility antigens with at least fourcommon allotypes from a given mammalian species.
 2. The composition ofclaim, wherein said allotypes each are present in 80% or more ofindividuals.
 3. The composition of claim 1, wherein any given cellexpresses only a single allotype.
 4. The composition of claim 1, whereinat least one cell expresses at least two allotypes.
 5. The compositionof claim 1, wherein said antigens are Class I antigens.
 6. Thecomposition of claim 1, wherein said antigens are Class II antigens. 7.The composition of claim 1, wherein said antigens are both Class I andClass II antigens or other alloantigens coded by polymorphic genes. 8.The composition of claim 1, wherein said plurality is representative ofall known allotypes of said mammalian species.
 9. The composition ofclaim 1, wherein said mammal is a human.
 10. The composition of claim 9,wherein said allotypes include at least one of the following humanallotypes: HLAA₁, A₂, A₃, A₁₁, A₂₄, A₂₉, A₃₂, B₇, B₈, B₁₃, B₃₅, B₃₈,B₄₄, B₅₅, B₆₀, B₆₂, CW₁, CW₂, CW₄, CW₅, CW₆, CW₇, CW₉, CW₁₀, CW₁₁, DR₁,DR₃, DR₄, DR₇, DR₈, DR₁₁, DR₁₂, DR₁₃, DR₁₅, ABO Blood Groups
 11. Thecomposition of claim 1, wherein said cells further express an antigenfrom an enveloped virus.
 12. The composition of claim 11, wherein saidvirus is a herpesvirus.
 13. The composition of claim 12, wherein saidvirus is a retrovirus.
 14. The composition of claim 1, wherein saidintact cells further express a cytokine.
 15. The composition of claim14, wherein said cytokine is selected from the group consisting of IL-1,IL-2, IL-4, IL-7, IL-12, γ-interferon and GMCSF.
 16. The composition ofclaim 1, wherein said intact cells further express a costimulatorymolecule.
 17. The composition of claim 16, wherein said costimulatorymolecule is B-7.
 18. The composition of claim 1, wherein said cells arerendered incapable of growth.
 19. The composition of claim 18, whereinsaid cells are lethally irradiated.
 20. The composition of claim 1,further comprising a pharmaceutically acceptable carrier, diluent orexcipient.
 21. A method for generating an immune response in a givenmammal comprising: (a) providing a composition comprising (i) intactcells, wherein said cells express major histocompatibility antigens withat least four allotypes from the species of said given mammal; and (ii)a pharmaceutically acceptable carrier, diluent or excipient, (b)administering said composition to said given mammal.
 22. The method ofclaim 21, wherein said mammal is a human.
 23. The method of claim 21,wherein said intact cells further express an antigen from an envelopedvirus.
 24. The method of claim 21, wherein step (a) is followed, andstep (b) is preceded, by lethal irradiation of said cells.
 25. A methodfor eliciting an immune response in a given mammal against an envelopedvirus comprising: (a) identifying a given mammal at risk of infectionwith said virus; (b) providing a composition comprising (i) intactcells, wherein said cells express major histocompatibility antigens witha plurality of allotypes from the species of said given mammal; (ii) apharmaceutically acceptable carrier, diluent or excipient, (b)administering said composition to said given mammal in an amounteffective to elicit said immune response.
 26. The method of claim 25,wherein said mammal is a human.
 27. The method of claim 25, wherein step(a) is followed, and step (b) is preceded, by irradiation of said cells.28. The method of claim 25, wherein said intact cells further express anantigen from an enveloped virus.
 29. A composition comprising intact,non-malignant cells, wherein said cells express major histocompatibilityantigens with a plurality of allotypes from a given mammalian species.30. The composition of claim 1, further comprising at least onerecombinant major or minor allotypic antigen of said species.
 31. Thecomposition of claim 30, wherein said recombinant antigen is produced ina host selected from the group consisting of bacteria, fungi, insectcells and mammalian cells.